Reanalysis of "Hexamethylene amiloride synergizes with venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia." by Jiang, X. et al., iScience, 2024

Jiang, X., Huang, K., Sun, X., Li, Y., Hua, L., Liu, F., Huang, R., Du, J., & Zeng, H. (2024). Hexamethylene amiloride synergizes with venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia.. iScience, 27(1), 108691.

Abstract

In this study, Jiang et al. [1] profiled acute myeloid leukemia (AML) cell lines under treatment with Hexamethylene amiloride (HA) and venetoclax, alone and in combination, to further our understanding of the therapeutic potential of targeting NHE1 in AML. The reanalysis of this dataset includes a comprehensive RNA-seq analysis pipeline consisting of UMAP (Uniform Manifold Approximation and Projection) [2], PCA (Principal Component Analysis) [3], and t-SNE (t-distributed Stochastic Neighbor Embedding) [4] plots for visualizing sample distributions. A clustergram heatmap provides an overview of gene expression patterns across samples. Differential gene expression analysis is performed for each control and perturbation sample pair. Enrichment analysis for each resulting gene signature is conducted using Enrichr [5, 6, 7]. Transcription factor analysis of these gene signatures is performed utilizing ChEA3 [8]. Furthermore, the reanalysis incorporates reverser and mimicker drug match analysis using L2S2 [9] and DRUG-seqr [10], considering both FDA-approved and non-FDA-approved compounds. Results are presented as tables and bar charts.

This abstract was generated with the assistance of Gemini 2.0 Flash.

Methods

RNA-seq alignment

Gene count matrices were obtained from ARCHS4 [11], which preprocessed the raw FASTQ data using the Kallisto [12] and STAR [] pseudoalignment algorithm.

Gene matrix processing

The raw gene matrix was filtered to remove genes that do not have an average of 3 reads across the samples. It was then quantile, log2, and z-score normalized. A regex-based function was used to infer whether individual samples belong to a “control” or a “perturbation” group by processing the metadata associated with each sample.

Dimensionality Reduction Visualization

Three types of dimensionality reduction techniques were applied on the processed expression matrices: UMAP[2], PCA[3], and t-SNE[4]. UMAP was calculated by the UMAP Python package and PCA and t-SNE were calculated using the Scikit-Learn Python library. The samples were then represented on 2D scatterplots.

Clustergram Heatmap

As a preliminary step, the top 1000 genes exhibiting most variability were selected. Using this new set, clustergram heatmaps were generated. Two versions of the clustergram exist: an interactive one generated by Clustergrammer [13] and a publication-ready alternative.

Differentially Expressed Genes Calculation and Volcano Plot

Differentially expressed genes between the control and perturbation samples were calculated using Limma Voom [14]. The logFC and -log10p values of each gene were visualized as a volcano scatterplot. Upregulated and downregulated genes were selected according to this criteria: p < 0.05 and |logFC| > 1.0.

Enrichr Enrichment Analysis

The upregulated and down-regulated sets were separately submitted to Enrichr [5, 6, 7]. These sets were compared against libraries from ChEA [8], ARCHS4 [12], Reactome Pathways [15], MGI Mammalian Phenotype [16], Gene Ontology Biological Processes [17], GWAS Catalog [18], KEGG [19, 20, 21], and WikiPathways [22]. The top matched terms from each library and their respective -log10p values were visualized as barplots.

Chea3 Transcription Factor Analysis

The upregulated and down-regulated sets were separately submitted to Chea3 [8]. These sets were compared against the libraries ARCHS4 Coexpression [12], GTEx Coexpression [23], Enrichr [5, 6, 7], ENCODE ChIP-seq [24, 25], ReMap ChIP-seq [26], and Literature-mined ChIP-seq. The top matched TFs were ranked according to their average score across each library and represented as barplots.

L2S2 and Drug-seqr drug analysis

The top 500 up and downregulated sets were submitted simulataneously to identify reverser and mimicker molecules, both FDA and non-FDA approved, from the L2S2 [9] and Drug-seqr [10] databases. The top matched molecules were compiled into tables and visualized as barplots.

GSM7884755 GSM7884756 GSM7884757 GSM7884758 GSM7884759 GSM7884760 GSM7884761 GSM7884762 GSM7884763 GSM7884764 GSM7884765 GSM7884766
TSPAN6 0 0 0 0 0 2 1 0 0 0 0 2
TNMD 0 0 0 0 0 0 0 0 0 0 0 0
DPM1 1475 1322 1197 1580 1236 1349 1090 899 1048 1126 1334 1148
SCYL3 417 291 281 357 288 300 385 296 349 264 241 260
C1ORF112 368 304 305 524 310 390 374 255 270 142 176 128

table 1: This is a preview of the first 5 rows of the raw RNA-seq expression matrix from GSE247175.

Results

Dimensionality Reduction

UMAP

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Figure 1: This figure displays a 2D scatter plot of a UMAP decomposition of the sample data. Each point represents an individual sample, colored by its experimental group.

PCA

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Figure 2: This figure displays a 2D scatter plot of a PCA decomposition of the sample data. Each point represents an individual sample, colored by its experimental group.

t-SNE

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Figure 3: This figure displays a 2D scatter plot using a t-SNE decomposition of the sample data. Each point represents an individual sample, colored by its experimental group.

Clustergram Heatmaps

Figure 4: The figure contains an interactive heatmap displaying gene expression for each sample in the RNA-seq dataset. Every row of the heatmap represents a gene, every column represents a sample, and every cell displays normalized gene expression values. The heatmap additionally features color bars beside each column which represent prior knowledge of each sample, such as the tissue of origin or experimental treatment.

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Figure 5: this figure is a clustergram produced with the graphing library Plotly. It sacrifices some interactivity for a more polished look.

Differentially Expressed Genes Calculation and Volcano Plots

logFC AveExpr t P.Value adj.P.Val B
gene_symbol
SRGN 2.14 9.83 105.66 3.328260e-20 6.334484e-16 36.70
CHI3L1 3.15 9.74 101.23 5.753391e-20 6.334484e-16 36.07
FTH1 2.63 10.86 76.01 2.238429e-18 1.504113e-14 32.71
HSPA8 1.85 9.18 74.83 2.732266e-18 1.504113e-14 32.46
FTL 1.46 11.82 72.27 4.263324e-18 1.877568e-14 31.81

Table 2: This is a preview of the first 5 rows of the differentially expressed gene table calculated by Limma Voom.

logFC AveExpr t P.Value adj.P.Val B
gene_symbol
HSPA8 1.35 8.98 32.01 3.204121e-13 7.055474e-09 20.70
SCD 1.21 8.72 26.74 2.851548e-12 3.139554e-08 18.65
FADS2 1.32 7.59 18.41 2.516819e-10 2.792420e-07 14.26
SQLE 1.05 6.75 18.39 2.553503e-10 2.792420e-07 14.22
INSIG1 1.36 6.41 18.11 3.059481e-10 2.929121e-07 14.02

Table 3: This is a preview of the first 5 rows of the differentially expressed gene table calculated by Limma Voom.

logFC AveExpr t P.Value adj.P.Val B
gene_symbol
TFRC 0.99 8.48 28.64 2.873655e-12 1.581947e-08 18.67
TUBB4B 0.93 7.84 24.34 1.898124e-11 4.179669e-08 16.81
LPCAT1 0.88 7.85 24.04 2.193322e-11 4.390631e-08 16.67
SFPQ 0.62 9.47 20.80 1.164689e-10 1.349813e-07 15.02
MT-CO1 0.41 13.45 20.34 1.509704e-10 1.662185e-07 14.07

Table 4: This is a preview of the first 5 rows of the differentially expressed gene table calculated by Limma Voom.

dmso-vs-combo

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Figure 6: The figure contains an interactive scatter plot which displays the log2-fold changes and statistical significance of each gene calculated by performing a differential gene expression analysis for the comparison dmso-vs-combo. Every point in the plot represents a gene. Red points indicate significantly up-regulated genes, blue points indicate down-regulated genes.

dmso-vs-ha

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Figure 7: The figure contains an interactive scatter plot which displays the log2-fold changes and statistical significance of each gene calculated by performing a differential gene expression analysis for the comparison dmso-vs-ha. Every point in the plot represents a gene. Red points indicate significantly up-regulated genes, blue points indicate down-regulated genes.

dmso-vs-ven

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Figure 8: The figure contains an interactive scatter plot which displays the log2-fold changes and statistical significance of each gene calculated by performing a differential gene expression analysis for the comparison dmso-vs-ven. Every point in the plot represents a gene. Red points indicate significantly up-regulated genes, blue points indicate down-regulated genes.

Enrichr: Enrichment Analysis

Upregulated Set

dmso-vs-combo

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Figure 9: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=f46b595ce1c8abb5b5f47c6e078592d4

dmso-vs-ha

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Figure 10: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=9e29b635bff0f028bf85ef8e41c65a83

dmso-vs-ven

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Figure 11: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=372778e4f84d2a69f1b68d91c1f925a1

Downregulated Set

dmso-vs-combo

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Figure 12: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=372778e4f84d2a69f1b68d91c1f925a1

dmso-vs-ha

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Figure 13: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=372778e4f84d2a69f1b68d91c1f925a1

dmso-vs-ven

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Figure 14: This figure contains several barplots depicting enrichment analysis results on the upregulated gene set. Each barplot corresponds to an individual library from Enrichr, and the top matching terms by p-value are depicted in each. Statistically significant terms are represented as red bars while others are represented as gray. Access your Enrichment results here: https://amp.pharm.mssm.edu/Enrichr/enrich?dataset=372778e4f84d2a69f1b68d91c1f925a1

CHEA3: Transcription Factor Enrichment Analysis

Upregulated Set

dmso-vs-combo

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Figure 15: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

dmso-vs-ha

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Figure 16: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

dmso-vs-ven

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Figure 17: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

Downregulated Set

dmso-vs-combo

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Figure 18: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

dmso-vs-ha

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Figure 19: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

dmso-vs-ven

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Figure 20: Horizontal bar chart, y-axis represents transcription factors. Displays the top ranked transcription factors for the upregulated set according to their average integrated scores across all the libraries.

L2S2 and DRUG-seqr: Reverser and Mimicker Drugs

Reverser Results

dmso-vs-combo

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueReverse adjPvalueReverse oddsRatioReverse reverserOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 8.99e-01 1 7.01e-01 8 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.00e+00 1 0.00e+00 0 False 712

Table 5: Ranked LINCS L1000 signatures predicted to reverse the uploaded geneset.

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Figure 21: barplot representation depicting the -log10p values of the top l2s2_all reversers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

dmso-vs-ha

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueReverse adjPvalueReverse oddsRatioReverse reverserOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 8.99e-01 1 7.01e-01 8 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.00e+00 1 0.00e+00 0 False 712

Table 6: Ranked LINCS L1000 signatures predicted to reverse the uploaded geneset.

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Figure 22: barplot representation depicting the -log10p values of the top l2s2_all reversers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

dmso-vs-ven

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueReverse adjPvalueReverse oddsRatioReverse reverserOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 8.99e-01 1 7.01e-01 8 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.00e+00 1 0.00e+00 0 False 712

Table 7: Ranked LINCS L1000 signatures predicted to reverse the uploaded geneset.

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Figure 23: barplot representation depicting the -log10p values of the top l2s2_all reversers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

Mimicker Results

dmso-vs-combo

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueMimic adjPvalueMimic oddsRatioMimic mimickerOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 3.77e-09 6.33e-03 3.24e+00 35 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.34e-08 1.13e-02 3.14e+00 34 False 712

Table 8: Ranked LINCS L1000 signatures predicted to mimic the uploaded geneset.

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Figure 24: barplot representation depicting the -log10p values of the top l2s2_all mimickers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

dmso-vs-ha

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueMimic adjPvalueMimic oddsRatioMimic mimickerOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 3.77e-09 6.33e-03 3.24e+00 35 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.34e-08 1.13e-02 3.14e+00 34 False 712

Table 9: Ranked LINCS L1000 signatures predicted to mimic the uploaded geneset.

No description has been provided for this image

Figure 25: barplot representation depicting the -log10p values of the top l2s2_all mimickers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

dmso-vs-ven

l2s2_fda

Caught error:  No Results for l2s2_fda

l2s2_all

perturbation term pvalueMimic adjPvalueMimic oddsRatioMimic mimickerOverlap approved count
0 SL-0101-1 AICHI002_K562_4H_D11_SL-0101-1_0.04uM up 3.77e-09 6.33e-03 3.24e+00 35 False 192
1 GW-5074 AICHI001_THP1_4H_N14_GW-5074_2.5uM up 1.34e-08 1.13e-02 3.14e+00 34 False 712

Table 10: Ranked LINCS L1000 signatures predicted to mimic the uploaded geneset.

No description has been provided for this image

Figure 26: barplot representation depicting the -log10p values of the top l2s2_all mimickers. Red bars represent statistically significant results; otherwise gray.

drugseqr_fda

Caught error:  No Results for drugseqr_fda

drugseqr_all

Caught error:  No Results for drugseqr_all

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